Genetically modified tumor-targeted bacteria with reduced virulence

ABSTRACT

The present invention is directed to mutant  Salmonella  sp. having a genetically modified msbB gene in which the mutant  Salmonella  is capable of targeting solid tumors. The invention is also directed to  Salmonella  sp. containing a genetically modified msbB gene as well as an genetic modification in a biosynthetic pathway gene such as the purl gene. The present invention further relates to the therapeutic use of the mutant  Salmonella  for growth inhibition and/or reduction in volume of solid tumors.

This application is a continuation-in-part of application Ser. No.08/926,636, filed Sep. 10, 1997, the entire disclosure of which isincorporated by reference herein in its entirety.

1. FIELD OF THE INVENTION

The present invention is concerned with the isolation of a gene ofSalmonella which, when genetically disrupted, reduces both virulence andseptic shock caused by this organism and increases sensitivity to agentswhich promote eradication of the bacteria, e.g., chelating agents. Thenucleotide sequence of this gene and the means for its geneticdisruption are provided, and examples of the use of tumor-targetedbacteria which possess a disruption in this gene to inhibit growth ofcancers, including, but not limited to, melanoma, colon cancer, andother solid tumors are described. The present invention also providesfor the genetic disruption of this gene in combination with disruptionof an auxotrophic gene.

2. BACKGROUND OF THE INVENTION

Citation or identification of any reference in Section 2, or any sectionof this application shall not be construed as an admission that suchreference is available as prior art to the present invention.

A major problem in the chemotherapy of solid tumor cancers is deliveryof therapeutic agents, such as drugs, in sufficient concentrations toeradicate tumor cells while at the same time minimizing damage to normalcells. Thus, studies in many laboratories are directed toward the designof biological delivery systems, such as antibodies, cytokines, andviruses for targeted delivery of drugs, pro-drug converting enzymes,and/or genes into tumor cells. Houghton and Colt, 1993, New Perspectivesin Cancer Diagnosis and Management 1: 65-70; de Palazzo, et al., 1992a,Cell. Immunol. 142:338-347; de Palazzo et al., 1992b, Cancer Res. 52:5713-5719; Weiner, et al., 1993a, J. Immunotherapy 13:110-116; Weiner etal., 1993b, J. Immunol. 151:2877-2886; Adams et al., 1993, Cancer Res.53:4026-4034; Fanger et al., 1990, FASEB J. 4:2846-2849; Fanger et al.,1991, Immunol. Today 12:51-54; Segal, et al., 1991, Ann N.Y. Acad. Sci.636:288-294; Segal et al., 1992, Immunobiology 185:390-402; Wunderlichet al., 1992; Intl. J. Clin. Lab. Res. 22:17-20; George et al., 1994, J.Immunol. 152:1802-1811; Huston et al., 1993, Intl. Rev. Immunol.10:195-217; Stafford et al., 1993, Cancer Res. 53:4026-4034; Haber etal., 1992, Ann. N.Y. Acad. Sci. 667:365-381; Haber, 1992, Ann. N.Y.Acad. Sci. 667: 365-381; Feloner and Rhodes, 1991, Nature 349:351-352;Sarver and Rossi, 1993, AIDS Research & Human Retroviruses 9:483-487;Levine and Friedmann, 1993, Am. J. Dis. Child 147:1167-1176; Friedmann,1993, Mol. Genetic Med. 3:1-32; Gilboa and Smith, 1994, Trends inGenetics 10:139-144; Saito et al., 1994, Cancer Res. 54:3516-3520; Li etal., 1994, Blood 83:3403-3408; Vieweg et al., 1994, Cancer Res.54:1760-1765; Lin et al., 1994, Science 265:666-669; Lu et al., 1994,Human Gene Therapy 5:203-208; Gansbacher et al., 1992, Blood80:2817-2825; Gastl et al., 1992, Cancer Res. 52:6229-6236.

2.1 Bacterial Infections and Cancer

Regarding bacteria and cancer, an historical review reveals a number ofclinical observations in which cancers were reported to regress inpatients with bacterial infections. Nauts et al., 1953, Acta Medica.Scandinavica 30 145:1-102, (Suppl. 276) state:

-   -   The treatment of cancer by injections of bacterial products is        based on the fact that for over two hundred years neoplasms have        been observed to regress following acute infections, principally        streptococcal. If these cases were not too far advanced and the        infections were of sufficient severity or duration, the tumors        completely disappeared and the patients remained free from        recurrence.

Shear, 1950, J. A.M.A. 142:383-390 (Shear), observed that 75 percent ofthe spontaneous remissions in untreated leukemia in-the Children'sHospital in Boston occurred following an acute episode of bacterialinfection. Shear questioned:

-   -   Are pathogenic and non-pathogenic organisms one of Nature's        controls of microscopic foci of malignant disease, and in making        progress in the control of infectious diseases, are we removing        one of Nature's controls of cancer?

Subsequent evidence from a number of research laboratories indicatedthat at least some of the anti-cancer effects are mediated throughstimulation of the host immune system, resulting in enhancedimmuno-rejection of the cancer cells. For example, release of thelipopolysaccharide (LPS) endotoxin by gram-negative bacteria such asSalmonella triggers release of tumor necrosis factor, TNF, by cells ofthe host immune system, such as macrophages, Christ et al., 1995,Science 268:80-83. Elevated TNF levels in turn initiate a cascade ofcytokine-mediated reactions which culminate in the death of tumor cells.In this regard, Carswell et al., 1975, Proc. Natl. Acad. Sci. USA72:3666-3669, demonstrated that mice injected with bacillusCalmette-Guerin (BCG) have increased serum levels of TNF and thatTNF-positive serum caused necrosis of the sarcoma Meth A and othertransplanted tumors in mice. Further, Klimpel et al., 1990, J. Immunol.145:711-717, showed that fibroblasts infected in vitro with Shigella orSalmonella had increased susceptibility to TNF.

As a result of such observations as described above, immunization ofcancer patients with BCG injections is currently utilized in some cancertherapy protocols. See Sosnowski, 1994, Compr. Ther. 20:695-701; Barthand Morton, 1995, Cancer 75 (Suppl. 2):726-734; Friberg, 1993, Med.Oncol. Tumor. Pharmacother. 10:31-36 for reviews of BCG therapy.

2.2 Parasites and Cancer Cells

Although the natural biospecificity and evolutionary adaptability ofparasites has been recognized for some time and the use of theirspecialized systems as models for new therapeutic procedures has beensuggested, there are few reports of, or proposals for, the actual use ofparasites as vectors.

Lee et al., 1992, Proc. Natl. Acad. Sci. USA 89:1847-1851 (Lee et al.)and Jones et al., 1992, Infect. Immun. 60:2475-2480 (Jones et al.)isolated mutants of Salmonella typhimurium that were able to invadeHEp-2 (human epidermoid carcinoma) cells in vitro in significantlygreater numbers than the wild type strain. The “hyperinvasive” mutantswere isolated under conditions of aerobic growth of the bacteria thatnormally repress the ability of wild type strains to invade HEp-2 animalcells. However, Lee et al. and Jones et al. did not suggest the use ofsuch mutants as therapeutic vectors, nor did they suggest the isolationof tumor-specific bacteria by selecting-for mutants that show infectionpreference for melanoma or other cancers over normal cells of the body.Without tumor-specificity or other forms of attenuation, suchhyperinvasive Salmonella typhimurium as described by Lee et al. andJones et al. would likely be pan-invasive, causing wide-spread infectionin the cancer patient.

2.3 Tumor-Targeted Bacteria

Genetically engineered Salmonella have been demonstrated to be capableof tumor targeting, possess anti-tumor activity and are useful indelivering effector genes such as the herpes simplex thymidine kinase(HSV TK) to solid tumors (Pawelek et al., WO 96/40238). Two significantconsiderations for the in vivo use of bacteria are their virulence andability to induce tumor necrosis factor a (TNFα)-mediated septic shock.As TNFα-mediated septic shock is among the primary concerns associatedwith bacteria, modifications which reduce this form of an immuneresponse would be useful because TNFα levels would not become toxic, anda more effective concentration and/or duration of the therapeutic vectorcould be used.

2.4 Modified Bacterial Lipid A

Modifications to the lipid composition of tumor-targeted bacteria whichalter the immune response as a result of decreased induction of TNFαproduction were suggested by Pawelek et al. (Pawelek et al., WO96/40238). Pawelek et al. provided methods for isolation of genes fromRhodobacter responsible for monophosphoryl lipid A (MLA) production. MLAacts as an antagonist to septic shock. Pawelek et al. also suggested theuse of genetic modifications in the lipid A biosynthetic pathway,including the mutation firA, which codes for the third enzyme UDP-3-O(R-30 hydroxylmyristoly)-glucosamine N-acyltransferase in lipid Abiosynthesis (Kelley et al., 1993, J. Biol. Chem. 268: 19866-19874).Pawelek et al. showed that mutations in the firA gene induce lowerlevels of TNFα. However, these authors did not suggest enzymes whichmodify the myristate portion of the lipid A molecule. Furthermore,Pawelek et al. did not suggest that modifications to the lipid contentof bacteria would alter their sensitivity to certain agents, such aschelating agents.

In Escherichia coli, the gene msbB (mlt) which is responsible for theterminal myristalization of lipid A has been identified (Engel, et al.,1992, J. Bacteriol. 174:6394-6403; Karow and Georgopoulos 1992, J.Bacteriol. 174: 702-710; Somerville et al., 1996, J. Clin. Invest. 97:359-365). Genetic disruption of this gene results in a stablenon-conditional mutation which lowers TNFα induction (Somerville et al.,1996, J. Clin. Invest. 97: 359-365). These references, however, do notsuggest that disruption of the msbB gene in tumor-targeted Salmonellavectors would result in bacteria which are less virulent and moresensitive to chelating agents.

The problems associated with the use of bacteria as gene deliveryvectors center on the general ability of bacteria to directly killnormal mammalian cells as well as their ability to overstimulate theimmune-system via TNFα which can have toxic consequences for the host(Bone, 1992 JAMA 268: 3452-3455; Dinarello et al., 1993 JAMA 269:1829-1835). In addition to these factors, resistance to antibiotics canseverely complicate coping with the presence of bacteria within thehuman body (Tschape, 1996, D T W Dtsch Tierarztl Wochenschr 1996103:273-7; Ramos et al., 1996, Enferm Infec. Microbiol. Clin. 14:345-51).

Hone and Powell, WO97/18837 (“Hone and Powell”), disclose methods toproduce gram-negative bacteria having non-pyrogenic Lipid A or LPS.Although Hone and Powell broadly asserts that conditional mutations in alarge number of genes including msbB, kdsA, kdsB, kdtA, and htrB, etc.can be introduced into a broad variety of gram-negative bacteriaincluding E. coli, Shigella sp., Salmonella sp., etc., the only mutationexemplified is an htrB mutation introduced into E. coli. Further,although Hone and Powell propose the therapeutic use of non-pyrogenicSalmonella with a mutation in the msbB gene, there is no enablingdescription of how to accomplish such use. Moreover, Hone and Powellpropose using non-pyrogenic bacteria only for vaccine purposes.

The objective of a vaccine vector is significantly different from thepresently claimed tumor-targeted vectors. Thus, vaccine vectors haverequirements quite different from tumor-targeted vectors. Vaccinevectors are intended to elicit an immune response. A preferred livebacterial vaccine must be immunogenic so that it elicits protectiveimmunity; however, the vaccine must not be capable of excessive growthin vivo which might result in adverse reactions. According to theteachings of Hone and Powell, a suitable bacterial vaccine vector istemperature sensitive having minimal replicative ability at normalphysiological ranges of body temperature.

In contrast, preferred tumor-targeted parasitic vectors, such as but notlimited to Salmonella, are safely tolerated by the normal tissues of thebody such that pathogenesis is limited, yet the vectors target to tumorsand freely replicate within them. Thus, vaccine vectors which replicateminimally at normal body temperatures, would not be suitable for use astumor-targeted vectors.

The preferred properties of tumor-specific Salmonella strains include 1)serum resistance, allowing the parasite to pass through the vasculatureand lymphatic system in the process of seeking tumors, 2) facultativeanaerobiasis, i.e., ability to grow under anaerobic or aerobicconditions allowing amplification in large necrotic tumors which arehypoxic as well as small metastatic tumors which may be more aerobic, 3)susceptibility to the host's defensive capabilities, limitingreplication in normal tissues but not within tumors where the hostdefensive capabilities may be impaired, 4) attenuation of virulence,whereby susceptibility to the host defenses may be increased, and theparasite is tolerated by the-host, but does not limit intratumoralreplication, 5) invasive capacity towards tumor cells, aiding in tumortargeting and anti-tumor activity, 6) motility, aiding in permeationthroughout the tumor, 7) antibiotic sensitivity for control duringtreatment and for post treatment elimination (e.g., sensitivity toampicillin, chloramphenicol, gentamicin, ciprofloxacin), and lackingantibiotic resistance markers such as those used in strain construction,and 8) low reversion rates of phenotypes aiding in the safety to therecipient individual.

3. SUMMARY OF THE INVENTION

The present invention provides a means to enhance the safety oftumor-targeted bacteria, for example, by genetic modification of thelipid A molecule. The modified tumor-targeted bacteria of the presentinvention induce TNFα less than the wild type bacteria and have reducedability to directly kill normal mammalian cells or cause systemicdisease compared to the wild type strain. The modified tumor-targetedbacteria of the present invention have increased therapeutic efficacy,i.e., more effective dosages of bacteria can be used and for extendedtime periods due to the lower toxicity in the form of less induced TNFαand systemic disease.

The present invention provides compositions and methods for the geneticdisruption of the msbB gene in bacteria, such as Salmonella, whichresults in bacteria, such as Salmonella, possessing a lesser ability toelicit TNFα and reduced virulence compared to the wild type. In oneembodiment, the invention provides for improved methods for selectinggenetic disruptions of the msbB gene. Additionally, the geneticallymodified bacteria have increased sensitivity to a chelating agentcompared to bacteria with the wild type msbB gene. In a preferredembodiment, Salmonella having a disrupted msbB gene, which arehyperinvasive to tumor tissues, are able to replicate within the tumors,and are useful for inhibiting the growth and/or reducing the tumorvolume of sarcomas, carcinomas, lymphomas or other solid tumor cancers,such as germ line tumors and tumors of the central nervous system,including, but not limited to, breast cancer, prostate cancer, cervicalcancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer,thyroid cancer, astrocytoma, glioma, pancreatic cancer, stomach cancer,liver cancer, colon cancer, and melanoma.

In an embodiment of the present invention, the bacteria are attenuatedby other means, including but not limited biosynthetic pathway mutationsleading to auxotrophy. In one specific embodiment, the biosyntheticpathway mutation is a genetic disruption of the purI gene. In anotherembodiment, the bacteria express pro-drug converting enzymes includingbut not limited to HSV-TK, cytosine deaminase (CD), and p450oxidoreductase.

The present invention also provides a means for enhanced sensitivity foruse in terminating therapy and for post therapy elimination. Accordingto one embodiment of the present invention, the tumor-targeted bacteriahaving a genetically modified lipid A also have enhanced susceptibilityto certain agents, e.g., chelating agents. It is a further advantage tomodify tumor-targeted bacteria in this way because it increases theability to eliminate the bacteria with agents which have anantibiotic-like effect, such as chelating agents including, but notlimited to, Ethylenediaminetetraacetic Acid (EDTA), EthyleneGlycol-bis(β-aminoethyl Ether) N,N,N′,N′,-Tetraacetic Acid (EGTA), andsodium citrate. Modification to enhance the ability to eliminate thebacteria via exogenous means, such as the administration of an agent towhich the genetically modified bacteria are more sensitive than theirwild type counterparts, is therefore useful.

The present invention further provides for a Salmonella straincomprising deletion mutations in both the msbB gene as well as anauxotrophic gene. In a specific embodiment, the auxotrophic deletionmutation affects the purl gene. In a preferred embodiment, thesemutations lead to increased safety of the strain. In another preferredembodiment, the strain also carries other mutations described hereinwhich increase efficacy of the strain but are not essential for itssafety.

4. DEFINITIONS

As used herein, Salmonella encompasses all Salmonella species,including: Salmonella typhi, Salmonella choleraesuis, and Salmonellaenteritidis. Serotypes of Salmonella are also encompassed herein, forexample, typhimurium, a subgroup of Salmonella enteritidis, commonlyreferred to as Salmonella typhimurium.

Attenuation: Attenuation is a modification so that a microorganism orvector is less pathogenic. The end result of attenuation is that therisk of toxicity as well as other side-effects is decreased, when themicroorganism or vector is administered to the patient.

Virulence: Virulence is a relative term describing the general abilityto cause disease, including the ability to kill normal cells or theability to elicit septic shock (see specific definition below).

Septic shock: Septic shock is a state of internal organ failure due to acomplex cytokine cascade, initiated by TNFα. The relative ability of amicroorganism or vector to elicit TNFα is used as one measure toindicate its relative ability to induce septic shock.

Chelating agent sensitivity: Chelating agent sensitivity is defined asthe effective concentration at which bacteria proliferation is affected,or the concentration at which the viability of bacteria, as determinedby recoverable colony forming units (c.f.u.), is reduced.

5. BRIEF DESCRIPTION OF THE FIGURES

The present invention may be understood more fully by reference to thefollowing detailed description, illustrative examples of specificembodiments and the appended figures.

FIG. 1. The complete DNA sequence of the Salmonella wild type (WT) 14028msbB gene (SEQ ID NO:1) and the deduced amino acid sequence of theencoded protein (SEQ ID NO:2).

FIG. 2A-2C. Knockout construct generated using the cloned Salmonella WT14028 msbB gene. The cloned gene was cut with SphI and MluI therebyremoving approximately half of the msbB coding sequence, and thetetracycline resistance gene (TET) from pBR322 cut with AatII and AvaIwas inserted after blunt-ending using the Klenow fragment of DNApolymerase I. A=Knockout construct. B=Salmonella chromosomal copy ofmsbB. C=Salmonella disrupted chromosomal copy of msbB after homologousrecombination. The start codon (ATG) and stop codon (TAA) andrestriction sites AseI, BamHI, SphI, MluI, and EcoRV are shown. Theposition of two primers, P1 and P2 which generate two different sizedPCR products for either wild type or disrupted msbB are shown.

FIG. 3A-3C. Southern blot analysis of chromosomally disrupted SalmonellaWT 14028 msbB. A) Southern blot probed with the tetracycline gene,demonstrating its presence in the plasmid construct and the two clones,and its absence in the WT 14028 bacteria. B) Southern blot of a similargel probed with an ³²P-labeled AseI/BamHI fragment derived from thecloned msbB. The AseI enzyme cuts upstream of msbB, and the BamHI cutsin one location in the wild type, but in a second location in thetetracycline gene which results in a higher molecular weight product.Lane 1 (KO) shows the position of the band in the knockout construct,compared to the WT 14028 in lane 2 (WT). Lanes 3 and 4 show the clonesYS8211 and YS861 with a higher molecular weight product. C) Southernblot of a similar gel probed with an ³²P-labeled mluI fragment derivedfrom the cloned msbB. See text Section 7.2 for details.

FIG. 4. TNFα induction by live Salmonella WT 14028 in mice. 1×10⁸ livebacteria in 0.1 cc phosphate buffered saline of the wild type or msbB⁻disrupted strains were injected i.v. in the tail vein of Balb/c mice.The bar graph indicates the TNFα induction with error bars. Clone YS8211induces TNFα 32% compared to Salmonella WT 14028.

FIG. 5. TNFα response by Sinclair swine to live Salmonella WT 14028 andmsbB⁻ clone YS8212. TNFα levels were measured at 1.5 and 6.0 hoursfollowing i.v. introduction of 1×10⁹ c.f.u. Salmonella WT 14028 andYS8212. At 1.5 hours TNFα response was significantly lower (p≦0.011) inthe msbB deletion mutant compared to the wild type.

FIG. 6A-6B. Respiratory level changes induced by LPS from WT 14028 andmsbB⁻ clone YS8212. Sinclair swine were injected with A) 5 μg/kgpurified LPS or B) 500 μg/kg purified LPS and respiration rate wasdetermined. The 500 μg/kg of LPS from Salmonella WT 14028 raised therate of respiration to more than 4 times normal, whereas the rate ofrespiration in msbB⁻ LPS-treated animals was less than doubled.

FIG. 7. TNFα induction by live Salmonella WT 14028 in human monocytes.Human monocytes isolated from peripheral blood were exposed toincreasing amounts of Salmonella c.f.u. At 1.0×10⁵ c.f.u.,concentrations of TNFα induced by WT 14028 were more than 3 times higherthan those induced by a number of msbB⁻ clones, i.e., YS8211, YS8212,YS8658, and YS1170.

FIG. 8. TNFα production by human monocytes. Human monocytes isolatedfrom peripheral blood were exposed to increasing amounts of purifiedLPS. As little as 1 nanogram of LPS from wild type was sufficient toelicit a measurable TNFα response and was maximal at 10 ng. In contrast,100 μg of LPS from each of a number of msbB⁻ clones was insufficient togenerate any response. Thus, at 10 ng LPS, the concentration of TNFαinduced by Salmonella WT 14028 was at least 10⁵ times higher thanconcentrations of TNFα induced by the independent msbB knockouts, i.e.,YS7216 and YS8211, and the derivatives, i.e., YS1170, YS8644, YS1604,YS8212, YS8658, YS1601, YS1629.

FIG. 9A-9B. Survival of mice and Sinclair swine, injected with 2×10⁷ or1×10⁹ respectively of live bacteria. A) WT 14028 killed all the mice in4 days, whereas the msbB⁻ clone YS862 spared 90% of the mice past 20days. B) Similarly, WT 14028 killed all the swine in 3 days, whereas themsbB⁻ clone YS8212 spared 100% of the swine past 20 days.

FIG. 10. Biodistribution of msbB⁻ Salmonella YS8211 in B16F10 melanomatumors. At 5 days, the ratio of msbB⁻ Salmonella within the tumorscompared to those in the liver exceeded 1000:1.

FIG. 11. Tumor retardation by msbB⁻ Salmonella. B16F10 melanoma tumorswere implanted in the flank of C57BL/6 mice and allowed to progress today 8. Mice either received no bacteria (control) or msbB⁻ strainsYS8211, YS8212, YS7216, YS1629. Two of the strains, YS8211 and YS1629retarded tumor progression significantly, whereas strains YS7216 andYS8212 did not.

FIG. 12A-12B. Sensitivity of WT 14028 and msbB disrupted bacteria tochelating agents. Wild type and msbB disrupted Salmonella clone YS8211and YS862 were grown in LB broth lacking sodium chloride (LB-zero), inthe presence or absence of 1 mM EDTA (FIG. 12A) or in the presence orabsence of 10 mM sodium citrate (FIG. 12B). The OD₆₀₀ was determined andplotted as a function of time. The msbB+ strain showed little inhibitionby EDTA or sodium citrate, compared to the msbB⁻ strains which showednear complete cessation of growth after 3 hours for EDTA or sodiumcitrate.

FIG. 13A-13B. Survival of msbB⁻ bacteria within murine macrophages.Murine bone marrow-derived macrophages (FIG. 13A) and a murinemacrophage cell line, J774, (FIG. 13B) were used as hosts for bacterialinternalization and quantified over time. The data are presented as apercentage of initial c.f.u.

FIG. 14. Conversion of msbB1(Δ)::tet to tet^(s) using the positiveselection suicide vector pCVD442 carrying a second version of the msbB⁻(msbB2(Δ)amp^(R) sacB⁺).

FIG. 15. Schematic diagram of the derivation of strain YS1456 from wildtype Salmonella typhimurium. See text Section 8.1 for details.

FIG. 16. Schematic diagram of the derivation of strain YS1646 from wildtype Salmonella typhimurium. See text Section 8.2 for details.

FIG. 17. Effect of YS1646 dose on B16-B10 murine melanoma tumor growth.

FIG. 18. Antibiotic suppression of YS1646-induced mortality followinglethal infection.

6. DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the isolation of a gene of Salmonella,i.e., msbB, which, when present in its normal form, contributes to TNFαinduction, general virulence, survival within macrophages, andinsensitivity to certain agents which promote eradication of thebacteria. The present invention is directed to the genetic modificationof the gene which results in disrupting the normal function of theproduct of the gene, and the incorporation of the genetic modificationinto tumor-targeted bacteria, including Salmonella, for therapeutic use.In a preferred embodiment, the bacteria have a genetic modification ofthe msbB gene as well as genetic modification of a gene in abiosynthetic pathway, such as the purl gene, resulting in an auxotrphicstrain.

In a preferred embodiment, the genetically modified bacteria are used inanimals, including humans, for reduction of volume and/or growthinhibition of solid tumors.

In an additional preferred embodiment, bacteria useful for the presentinvention show preference for attachment to and penetration into certainsolid tumor cancer cells or have an enhanced propensity to proliferatein tumor tissues as compared to normal tissues. These bacteria, includebut are not limited to Salmonella, having a natural ability todistinguish between cancerous or neoplastic cells tissues and normalcells/tissues.

Alternatively, tumor cell-specific bacteria useful for the invention maybe selected for and/or improved in tumor targeting ability using themethods described by Pawelek et al., WO 96/40238 incorporated herein byreference. Pawelek et al. describe methods for isolating tumorcell-specific bacteria by cycling a microorganism through pre-selectedtarget cells, preferably solid tumor cells in vitro, or through a solidtumor in vivo, using one or more cycles of infection.

6.1 Isolation/Identification of a Gene Involved in Virulence

The E. coli gene, msbB, has been shown to be involved in myristilizationof lipid A (Somerville et al., 1996, J. Clin. Invest. 97:359-365.) Thechromosomal organization of the E. coli msbB gene and the DNA sequencecoding for the msbB gene have been described (Engel, et al., 1992, J.Bacteriol. 174:6394-6403; Karow and Georgopoulos, 1992, J. Bacteriol.174: 702-710; Somerville et al., 1996, J. Clin. Invest. 97:-359-365).

As shown in the present invention, the msbB gene can be isolated frombacterial strains, other than E. coli, using low stringency DNA/DNAhybridization techniques known to those skilled in the art. (Sambrook etal., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989). Foran illustrative example of isolation of a msbB gene of bacteria,including but not limited to Salmonella spp., see Section 7.1 infra. Abacterial DNA library can be probed with a ³²P-labeled msbB gene from E.coli. Hybridizing clones are determined to be correct if they containDNA sequences similar to the known E. coli msbB gene.

6.1.1 Genetic Alteration of Salmonella msbB

One embodiment of the present invention provides a composition of matterwhich is a strain of bacteria with a genetic alteration in the msbBgene. In a preferred embodiment, the bacteria is Salmonella sp. Geneticalteration in the form of disruption or deletion can be accomplished byseveral means known to those skilled in the art, including homologousrecombination using an antibiotic resistance marker. These methodsinvolve disruption of the plasmid-based, cloned msbB gene usingrestriction endonucleases such that part or all of the gene is disruptedor eliminated or such that the normal transcription and translation areinterrupted, and an antibiotic resistance marker for phenotypicselection is inserted in the region of that deletion, disruption orother alteration. Linearized DNA is transformed into Salmonella, andbacteria bearing the antibiotic resistance are further examined forevidence of genetic alteration. Means for examining genetic alterationinclude PCR analysis and Southern blotting. For an illustrative exampleof genetic disruption of a Salmonella msbB gene, see Section 7.2.

In another embodiment of the invention, the msbB⁻/antibiotic resistancemarker can be transduced into a new bacterial strain. An illustrativeexample is provided in Section 7.2. Bacteriophage P22 and a SalmonellamsbB⁻ clone can be grown in zero salt Luria broth and the new phages inthe supernate can be used to infect a new Salmonella strain.

Yet another embodiment of the present invention provides Salmonella thatare attenuated in more than one manner, e.g., a mutation in the pathwayfor lipid A production, such as the msbB mutation described herein andone or more mutations to auxotrophy for one or more nutrients ormetabolites, such as uracil biosynthesis, purine biosynthesis, andarginine biosynthesis as described by Bochner, 1980, J. Bacteriol.143:926-933 herein incorporated by reference. In a preferred embodiment,the ability of msbB⁻ Salmonella to accumulate within tumors is retainedby msbB⁻ Salmonella having one or more mutations resulting in anauxotrophic strain. In a more preferred mode of this embodiment of theinvention, the bacterial vector which selectively targets tumors andexpresses a pro-drug converting enzyme is auxotrophic for uracil,aromatic amino acids, isoleucine and valine and synthesizes an alteredlipid A. In a specific preferred embodiment the msbB⁻ Salmonella alsocontain a genetic modification of the biosynthetic pathway gene, purI,leading to decreased virulence of the strain compared to wild type. Anillustrative example is provided in Sections 7 and 8.

6.1.2 Characteristics of Salmonella Having Disrupted msbB

A characteristic of the msbB⁻ Salmonella, described herein, is decreasedability to induce a TNFα response compared to the wild type bacterialvector. Both the whole bacteria and isolated or purifiedlipopolysaccharide (LPS) elicit a TNFα response. In an embodiment of theinvention, the msbB⁻ Salmonella induce TNFα expression at about 5percent to about 40 percent compared to the wild type Salmonella sp. (inother words, the msbB⁻ Salmonella induce TNFα expression at about 5percent to about 40 percent of the level induced by wild typeSalmonella, e.g., WT 14028.) In a preferred embodiment of the invention,the msbB⁻ Salmonella induce TNFα expression at about 10 percent to about35 percent of that induced by a wild type Salmonella sp. In anembodiment of the invention, purified LPS from msbB⁻ Salmonella inducesTNFα expression at a level which is less than or equal to 0.001 percentof the level induced by LPS purified from wild type Salmonella sp. TNFαresponse induced by whole bacteria or isolated or purified LPS can beassessed in vitro or in vivo using commercially available assay systemssuch as by enzyme linked immunoassay (ELISA). For illustrative examples,see sections 7.3.1 and 7.3.2 infra. comparison of TNFα production on aper c.f.u. or on a μg/kg basis, is used to determine relative activity.Lower TNFα levels on a per unit basis indicate decreased induction ofTNFα production.

Reduction of Virulence

Another characteristic of the msbB⁻ Salmonella, described herein, isdecreased virulence towards the host cancer patient compared to the wildtype bacterial vector. Wild type Salmonella can under some circumstancesexhibit the ability to cause significant progressive disease. Acutelethality can be determined for normal wild type live Salmonella andlive msbB⁻ Salmonella using animal models. For an illustrative example,see Section 7.4 and Section 9, Table III. Comparison of animal survivalfor a fixed inoculum is used to determine relative virulence. Strainshaving a higher rate of survival have decreased virulence.

Decreased Survival within Macrophages

Another characteristic of msbB⁻ Salmonella described herein, isdecreased survival within macrophage cells as compared to survival ofwild type bacteria. Wild type Salmonella (e.g., ATCC 14028) are notedfor their ability to survive within macrophages (Baumler, et al., 1994,Infect. Immun. 62:1623-1630; Buchmeier and Heffron 1989, Infect. Immun.57:1-7; Buchmeier and Heffron, 1990, Science 248:730-732; Buchmeier etal., 1993, Mol. Microbiol. 7:933-936; Fields et al., 1986, Proc. Natl.Acad. Sci. USA 83:5189-93; Fields et al., 1989, Science 243:1059-62;Fierer et al., 1993, Infect. Immun. 61:5231-5236; Lindgren et al., 1996,Proc. Natal. Acad. Sci. USA 3197-4201; Miller et al., 1989, Proc. Natl.Acad. Sci. USA 86:5054-5058; Sizemore et al., 1997, Infect. Immun.65:309-312).

A comparison of survival time in macrophages can be made using an invitro cell culture assay. A lower number of c.f.u. over time isindicative of reduced survival within macrophages. For an illustrativeexample, see Section 8 infra. As shown therein, using thegentamicin-based internalization assay and bone marrow-derived murinemacrophages or the murine macrophage cell line J774, a comparison ofsurvival of WT 14028 and msbB⁻ clone YS8211 was determined. In anembodiment of the invention, survival occurs at about 50 percent toabout 30 percent; preferably at about 30 percent to about 10 percent;more preferably at about 10 percent to about 1 percent of survival ofthe wild type stain.

Increased Sensitivity

Another characteristic of one embodiment of the msbB⁻ Salmonella,described herein, is increased sensitivity of the tumor-targetedbacteria to specific chemical agents which is advantageously useful toassist in the elimination of the bacteria after administration in vivo.Bacteria are susceptible to a wide range of antibiotic classes. However,it has surprisingly been discovered that certain Salmonella msbB⁻mutants encompassed by the present invention are sensitive to certainchemicals which are not normally considered antibacterial agents. Inparticular, certain msbB⁻ Salmonella mutants are more sensitive than WT14028 to chelating agents.

Previous descriptions of msbB⁻ E. coli have not suggested increasedsensitivity to such chelating agents. To the contrary, reports haveincluded increased resistance to detergents such as deoxycholate (Karowand Georgopoulos 1992 J. Bacteriol. 174: 702-710).

To determine sensitivity to chemical agents, normal wild type bacteriaand msbB⁻ bacteria are compared for growth in the presence or absence ofa chelating agent, for example, EDTA, EGTA or sodium citrate. Comparisonof growth is measured as a function of optical density, i.e., a loweroptical density in the msbB⁻ strain grown in the presence of an agent,than when the strain is grown in its absence, indicates sensitivity.Furthermore, a lower optical density in the msbB⁻ strain grown in thepresence of an agent, compared to the msbB⁺ strain grown in itspresence, indicates sensitivity specifically due to the msbB mutation.For an illustrative example, see section 7.7 infra. In an embodiment ofthe invention, 90 percent inhibition of growth of msbB⁻ Salmonella(compared to growth of wild type Salmonella sp.) occurs at about 0.25 mMEDTA to about 0.5 mM EDTA, preferably at about 99 percent inhibition atabout 0.25 mM EDTA to above 0.5 mM EDTA, more preferably at greater than99 percent inhibition at about 0.25 mM EDTA to about 0.5 mM EDTA.Similar range of growth inhibition is observed at similar concentrationsof EGTA.

Derivatives of msbB Mutants

When grown in Luria Broth (LB) containing zero salt, the msbB⁻ mutantsof the present invention are stable, i.e., produce few derivatives (asdefined below). Continued growth of the msbB⁻ mutants on modified LB (10g tryptone, 5 g yeast extract, 2 ml 1N CaCl₂, and 2 ml 1N MgSO₄ perliter, adjusted to pH 7 using 1N NaOH) also maintains stable mutants.

In contrast, when grown in normal LB, the msbB⁻ mutants may give rise toderivatives. As used herein, “derivatives” is intended to meanspontaneous variants of the msbB⁻ mutants characterized by a differentlevel of virulence, tumor inhibitory activity and/or sensitivity to achelating agent when compared to the original msbB⁻ mutant. The level ofvirulence, tumor inhibitory activity, and sensitivity to a chelatingagent of a derivative may be greater, equivalent, or less compared tothe original msbB⁻ mutant.

Derivatives of msbB⁻ strains grow faster on unmodified LB than theoriginal msbB⁻ strains. In addition, derivatives can be recognized bytheir ability to grow on MacConkey agar (an agar which contains bilesalts) and by their resistance to chelating agents, such as EGTA andEDTA. Derivatives can be stably preserved by cryopreservation at −70° C.or lyophilization according to methods well known in the art (Cryz etal., 1990, In New Generation Vaccines, M. M. Levine (ed.), MarcelDekker, New York pp. 921-932; Adams, 1996, In Methods in MolecularMedicine: Vaccine Protocols, Robinson et al. (eds), Humana Press, NewJersey, pp. 167-185; Griffiths, Id. pp. 269-288.)

Virulence is determined by evaluation of the administered dose at whichhalf of the animals die (LD₅₀). Comparison of the LD₅₀ of thederivatives can be used to assess the comparative virulence. Decrease inthe LD₅₀ of a spontaneous derivative as compared to its msbB⁻ parent,indicates an increase in virulence. In an illustrative example, thefaster-growing derivatives either exhibit the same level of virulence, agreater level of virulence, or a lower level of virulence compared totheir respective original mutant strains (see Section 9, Table III.) Inanother example, the ability of a derivative to induce TNFα remains thesame as the original mutant strain (see Section 7.3, FIG. 7).

In an illustrative example, the derivatives can either inhibit tumorgrowth more than or less than their respective original mutant strains(see Section 7.6, FIG. 11). It is demonstrated in Section 7.6 that theoriginal msbB⁻ mutant, YS8211, significantly inhibits tumor growthwhereas a derivative of this clone, YS8212, has less tumor growthinhibition activity. In contrast, the derivative, YS1629, exhibitsenhanced tumor growth inhibition activity compared to its parent msbB⁻clone, YS7216.

A derivative which is more virulent than its parent mutant but whichdoes induce TNFα at a lower level when compared to the wild type, i.e.,at a level of about 5 percent to about 40 percent of that induced by thewild type Salmonella, can be further modified to contain one or moremutations to auxotrophy. In an illustrative example, the YS1170derivative is mutated such that it is auxotrophic for one or morearomatic amino acids, e.g., aroA, and thus can be made less virulent andis useful according to the methods of the present invention. In anadditional illustrative example, genetic modifications of the purl gene(involved in purine biosynthesis) yield Salmonella strains that are lessvirulent than the parent strain. (See Sections 7 and 8).

Prior to use of a derivative in the methods of the invention, thederivative is assessed to determine its level of virulence, ability toinduce TNFα, ability to inhibit tumor growth, and sensitivity to achelating agent.

6.2 Use of Salmonella with Disrupted msbB for Tumor Targeting and inVivo Treatment of Solid Tumors

According to the present invention, the msbB⁻ mutant Salmonella areadvantageously used in methods to produce a tumor growth inhibitoryresponse or a reduction of tumor volume in an animal including a humanpatient having a solid tumor cancer. For such applications, it isadvantageous that the msbB⁻ mutant Salmonella possess tumor targetingability or target preferably to tumor cells/tissues rather than normalcells/tissues. Additionally, it is advantageous that the msbB⁻ mutantSalmonella possess the ability to retard or reduce tumor growth and/ordeliver a gene or gene product that retards or reduces tumor growth.Tumor targeting ability can be assessed by a variety of methods known tothose skilled in the art, including but not limited to cancer animalmodels.

For example, Salmonella with a msbB⁻ modification are assayed todetermine if they possess tumor targeting ability using the B16F10melanoma subcutaneous animal model. A positive ratio of tumor to liverindicates that the genetically modified Salmonella possesses tumortargeting ability. For an illustrative example, see Section 7.5.

Salmonella with the msbB modification can be assayed to determine ifthey possess anti-tumor ability using any of a number of standard invivo models, for example, the B16F10 melanoma subcutaneous animal model.By way of an illustrative example, and not by way of limitation, tumorsare implanted in the flanks of mice and staged to day 8 and thenbacterial strains are injected i.p. Tumor volume is monitored over time.Anti-tumor activity is determined to be present if tumors are smaller inthe bacteria-containing groups than in the untreated tumor-containinganimals. For an illustrative example, see section 7.6 infra.

The Salmonella of the present invention for in vivo treatment aregenetically modified such that, when administered to a host, thebacteria is less toxic to the host and easier to eradicate from thehost's system. The Salmonella are super-infective, attenuated andspecific for a target tumor cell. In a more preferred embodiment, theSalmonella may be sensitive to chelating agents having antibiotic-likeactivity.

In addition, the Salmonella used in the methods of the invention canencode “suicide genes”, such as pro-drug converting enzymes or othergenes, which are expressed and secreted by the Salmonella in or near thetarget tumor. Table 2 of Pawelek et al. WO96/40238 at pages 34-35presents an illustrative list of pro-drug converting enzymes which areusefully secreted or expressed by msbB⁻ mutant Salmonella for use in themethods of the invention. Table 2 and pages 32-35 are incorporatedherein by reference. The gene can be under the control of eitherconstitutive, inducible or cell-type specific promoters. See Pawelek etal. at pages 35-43, incorporated herein by reference, for additionalpromoters, etc. useful for mutant Salmonella for the methods of thepresent invention. In a preferred embodiment, a suicide gene isexpressed and secreted only when a Salmonella has invaded the cytoplasmof the target tumor cell, thereby limiting the effects due to expressionof the suicide gene to the target site of the tumor.

In a preferred embodiment, the Salmonella, administered to the host,expresses the HSV TK gene. Upon concurrent expression of the TK gene andadministration of ganciclovir to the host, the ganciclovir isphosphorylated in the periplasm of the microorganism which is freelypermeable to nucleotide triphosphates. The phosphorylated ganciclovir, atoxic false DNA precursor, readily passes out of the periplasm of themicroorganism and into the cytoplasm and nucleus of the host cell whereit incorporates into host cell DNA, thereby causing the death of thehost cell.

The method of the invention for inhibiting growth or reducing volume ofa solid tumor comprises administering to a patient having a solid tumor,an effective amount of an isolated mutant Salmonella sp. comprising agenetically modified msbB gene, said mutant being capable of targetingto the solid tumor when administered in vivo. The msbB⁻ mutantSalmonella may also express a suicide gene as described above.

In addition, in one embodiment the isolated Salmonella is analyzed forsensitivity to chelating agents to insure for ease in eradication of theSalmonella from the patient's body after successful treatment or if thepatient experiences complications due to the administration of theisolated Salmonella. Thus, if Salmonella is employed which is sensitiveto a chelating agent, at about 0.25 mM to about 1.0 mM of a chelatingagent such as EGTA, EDTA or sodium citrate can be administered to assistin eradication of the Salmonella after the anti-tumor effects have beenachieved.

When administered to a patient, e.g., an animal for veterinary use or toa human for clinical use, the mutant Salmonella can be used alone or maybe combined with any physiological carrier such as water, an aqueoussolution, normal saline, or other physiologically acceptable excipient.In general, the dosage ranges from about 1.0 c.f.u./kg to about 1×10¹⁰c.f.u./kg; optionally from about 1.0 c.f.u./kg to about 1×10⁸ c.f.u./kg;optionally from about 1×10² c.f.u./kg to about 1×10⁸ c.f.u./kg;optionally from about 1×10⁴ c.f.u./kg to about 1×10⁸ c.f.u./kg.

The mutant Salmonella of the present invention can be administered by anumber of routes, including but not limited to: orally, topically,injection including, but limited to intravenously, intraperitoneally,subcutaneously, intramuscularly, intratumorally, i.e., direct injectioninto the tumor, etc.

The following series of examples are presented by way of illustrationand not by way of limitation on the scope of the invention.

7. EXAMPLE Loss of Virulence, Reduced TNFα Stimulation, and IncreasedChelating Agent Sensitivity, by Disruption of the Salmonella msbB 7.1Isolation and Composition of Salmonella msbB Gene

A Salmonella genomic DNA library was first constructed. Wild typeSalmonella typhimurium (ATCC strain 14028) were grown overnight andgenomic DNA extracted according to the methods of Sambrook et al.(Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborPress, Cold Spring Harbor, 1989). Size-selected restrictionendonuclease-digested fragments ranging from 2 to 10 kB were generatedby time-limited digestion with Sau3A and selected by agarose gelelectrophoresis. These fragments were ligated into pBluescript SK- andtransformed to E. coli DH5α. Random analysis of clones revealed DNAinserts in ≧87%, with average size=5.1 Kb. The library consisted of1.4×10⁴ independent clones. In order to reduce the hybridization of theE. coli-originated msbB probe, to the 100% homologous chromosomal genein E. coli, the entire library was harvested from the petri dishes byflooding them with phosphate buffered saline and using a glass rod todislodge the colonies, and the resulting bacterial population wassubjected to a large-scale plasmid isolation, resulting in an amplifiedSalmonella library plasmid pool. This plasmid pool was then transformedto Salmonella LT2 YS5010, thereby eliminating the E. coli background.

A probe for msbB homologues was generated using a clone of the E. colimsbB gene (Karow and Georgopoulos 1992 J. Bacteriol. 174: 702-710) bydigesting E. coli with BglII/HincII and isolating a 600 bp fragmentwhich corresponds to a portion of the coding sequence. This fragment waslabeled using α³²P-dCTP and used to probe the Salmonella library atlow-stringency conditions consisting of 6×SSC, 0.1% SDS, 2× Denhardts,0.5% non-fat dry milk overnight at 55° C. Strongly hybridizing colonieswere purified, and plasmids extracted and subjected to restrictiondigestion and in situ gel hybridization under the same conditions usedfor colony hybridization (Ehtesham and Hasnain 1991 BioTechniques 11:718-721). Further restriction digests revealed a 1.5 kB fragment of DNAwhich strongly: hybridized with the probe and was sequenced at the YaleUniversity Boyer Center using fluorescent dye termination thermal cyclesequencing. Sequence analysis revealed that the 1.5 kb fragmentcontained an msbB homologue which apparently lacked an initiatingmethionine corresponding to that of the E. coli gene. A probe consistingof the 5′ region of this clone was generated by performing restrictiondigests using EcoRI/XbaI and again hybridizing to the library. Thecomplete nucleotide sequence of the Salmonella msbB gene (SEQ ID NO:1)and the deduced amino acid sequence of the encoded protein (SEQ ID NO:2)is shown in FIG. 1. The DNA homology of the putative Salmonella msbB andthe E. coli msbB is 75%. The protein homology is 98%, confirming thatthe cloned Salmonella gene is a bona fide msbB.

7.2 Genetic Alteration of Salmonella msbB

A knockout construct was generated using the cloned Salmonella msbBgene. The cloned gene was cut with SphI and MluI, thereby removingapproximately half of the msbB coding sequence, and the tetracyclineresistance gene from pBR322, cut with AatII and AvaI, was inserted afterblunt-ending using the Klenow fragment of DNA polymerase I (FIG. 2A-2C).The knockout disruption was accomplished by homologous recombinationprocedures (Russell et al., 1989, J. Bacteriol. 171:2609); the constructwas linearized using SacI and KpnI, gel purified and transfected toSalmonella LT2 YS501 by electroporation. Bacteria from thetransformation protocol were first selected on tetracycline plates, andsubsequently examined for the presence of plasmid-containingnon-chromosomal integrated contaminants by ampicillin resistance and thepresence of plasmids as determined by standard plasmid mini-preps(Titus, D. E., ed. Promega Protocols and Applications Guide, PromegaCorp, 1991). Bacterial colonies which were tetracycline resistant yetlacked plasmids were subjected to a PCR-based analysis of the structureof their msbB gene. PCR was used with primers which generate a fragmentinclusive of the region into which the tetracycline gene was inserted,where the forward primer was GTTGACTGGGAAGGTCTGGAG (SEQ ID NO:3),corresponding to bases 586 to 606, and the reverse primer wasCTGACCGCGCTCTATCGCGG (SEQ ID NO:4), corresponding to bases 1465 to 1485.Wild type Salmonella msbB+ results in an approximately 900 base pairproduct, whereas the disrupted gene with the tetracycline insert resultsin an approximately 1850 base pair product. Several clones were obtainedwhere only the larger PCR product was produced, indicating that thedisruption in the msbB gene had occurred.

Southern blot analysis was used to confirm the disruption of thechromosomal copy of Salmonella msbB. The plasmid-based knockoutconstruct (KO) was compared with genomic DNA prepared from wild type andputative disrupted msbB clones, YS82, YS86, YS8211 and YS861. The DNAwas double digested with AseI/BamHI and separated by agarose gelelectrophoresis on 0.9% or 1.2% agarose. Results of YS8211 and YS861 arepresented in FIG. 3A-3C. Similar gels were subjected to three separatecriteria: 3A) the presence of the tetracycline gene when probed with an³²P-labeled tetracycline gene fragment, 3B) Restriction fragment lengthwhen probed with an ³²P-labeled AseI/BamHI fragment derived from thecloned msbB and 3C) the presence or absence of the msbB mluI fragmentremoved in order to disrupt the msbB gene and insert the tetracyclinegene (FIG. 3A-3C). Since the mluI fragment was removed in order todisrupt the msbB gene and insert the tetracycline gene, it is expectedthat this probe would hybridize with the wild type FIG. 3C (lane 2 WT)but not the knockout construct (lane 1 KO), or the clones, (lanes 3 and4 YS8211 and YS821) thereby confirming the genetic alteration of themsbB gene. Each of the clones examined exhibited all of the expectedcriteria for an msbB gene deletion (knockout). These data furtherconfirm that msbB exists as a single copy in the wild type Salmonella,as no other hybridizing bands were observed when probed with a labeledoligonucleotide derived from the cloned DNA.

After the msbB mutation was confirmed, additional strains containing themsbB⁻ mutation were generated. The Salmonella strains used included WT14028 and YS72 (pur⁻ xyl⁻ hyperinvasive mutant from WT 14028; Pawelek etal., WO 96/40238). P22 transduction was used to generate YS8211(msbB::tet) using YS82 as a donor and YS861 and YS862 (msbB1::tet) usingYS86 as a donor; all with WT 14028 as recipient. YS7216 (msbB1::tet fromYS72) was generated by transduction using YS82 as a donor. Severalderivatives are encompassed by the present invention, including but notlimited to derivatives of YS8211 (YS8212, YS1170), YS862 (YS8644,YS8658), and YS7216 (YS1601, YS1604, YS1629). In a preferred embodiment,spontaneous derivatives grow somewhat faster on Luria agar compared toWT 14028 or msbB⁻ clones generated by transduction. msbB⁺ strains weregrown in LB broth or on LB plates containing 1.5% agar at 37° C. msbB⁻strains were grown in modified LB containing 10 g tryptone, 5 g yeastextract, 2 ml 1N CaCl₂ and 2 ml 1N MgSO₄ per liter, adjusted to pH 7using 1N NaOH. For transducing msbB1::tet, LB lacking NaCl was used,with 4 mg/l tetracycline. Liquid cultures were shaken at 225 rpm. Fortumor targeting 30 experiments, cells were diluted 1:100 in LB, grown toOD₆₀₀=0.8 to 1.0, washed in phosphate buffered saline (PBS), andresuspended in PBS.

7.2.1 An Improved Method for Selecting msbB Genetic Alterations byPre-Selection with Sucrose

An improved method for selecting msbB genetic alterations bypre-selection with sucrose has been discovered. This pre-selectionmethod is based on the selection of colonies that retain the sacB gene.The sacB gene is responsible for the conversion of sucrose into a toxicchemical, levan, that is lethal to the host cells, and can therefore beused to select for recombinants. Only those strains that undergodeletion of the sacB gene survive on medium containing sucrose andtherefore have the sucrose resistance property suc^(r). As describedbelow, pre-selecting of colonies that retain the sacB gene, eliminatedthe need for dilutions and comparison of sucrose⁽⁺⁾ vs. sucrose⁽⁻⁾colonies as performed in the normal sucrose selection.

The Normal Selection Procedure for the Sucrase System

E. coli SM10 λpir carrying a plasmid with the msbB(Δ) b1a and sacB geneswas used as a donor. The b1a gene for betalactamase confers resistanceto ampicillin. In the normal selection procedure, the donor strain wasmated using standard mating procedures, with a Salmonella strain intowhich the plasmid with msbB(Δ) b1a sacB was to be introduced. Since theSalmonella strain contained a second antibiotic resistance marker (e.g.,streptomycin resistance), the recombinant Salmonella clones were thenselected for dual resistance to ampicillin and streptomycin. To test forresolution of an individual clone, dilutions of each clone were platedon LB lacking sucrose, or LB containing 5% sucrose. Only those strainsthat underwent deletion or alteration of the sacB gene survive onsucrose. Comparison of the number of clones on sucrose⁽⁺⁾ or sucrose⁽⁻⁾plates, indicates the fraction of bacterial cells that underwentresolution. Sucrose resistant colonies were then further tested forsensitivity to ampicillin and tetracycline. Tet^(s) and amp^(s)indicated excision of the sacB and b1a genes during cross-over with thepartial msbB gene region. PCR was then used to confirm the msbB isoformpresent in the tet^(s) amp^(s) clones.

Pre-Selection Protocol for the Sucrase System

A variation in the normal sucrase protocol allowed for the screening ofincreased numbers of colonies, by pre-selecting colonies that retain thesacB gene. This pre-selection method eliminated the need for examinationand comparison of sucrose⁽⁺⁾ vs. sucrose⁽⁻⁾ from a large number ofcolonies. After the conjugation procedure described above, the colonies(impure at this stage) were gridded directly to LB plates containing 5%sucrose and grown at 30° C. The resulting impure colonies, whichcontinued to grow, gave rise to survivors on sucrose. Of the sucroseresistant colonies, those which displayed a phenotypic variation of“fuzzy edges” were then subjected to dilution and plated on sucrose (+)or sucrose (−) plates. Colonies were then tested for sensitivity totetracycline and ampicillin as above, and the msbB isoform was confirmedby PCR. This improved method was used to generate strains for P22 phagetransduction of msbB(A) b1a sacB chromosomal element. These strains werethen used to generate the YS1456 and YS1646 stains, which representpreferred embodiments of the novel msbB mutations of the presentinvention (see FIGS. 15 and 16).

7.3 Disruption of Salmonella msbB Reduced TNFα Induction

7.3.1 TNFα Induction in Mice

WT 14028 and the msbB⁻ clone YS8211, were first grown to saturation inLB media at 37° C. with shaking at 225 rpm. A 1:100 dilution of thesebacterial strains were then transferred to fresh LB and grown to anOD₆₀₀=1.0 at 37° C. with shaking at 225 rpm. The bacteria were dilutedin phosphate buffered saline and 1.0×10⁸ c.f.u. (about 5×10⁹ c.f.u./kg)were injected into the tail vein of Balb/C mice (n=4/strain), with PBSas a negative control. After 1.5 hours, serum was harvested intriplicate samples by cardiac puncture, centrifuged to remove thecellular content, and analyzed for TNFα using a Biosource InternationalCytoscreen ELISA plate, which was read on a Molecular Devices Emaxmicroplate reader.

Results are presented in FIG. 4 and expressed as a percent of the levelof TNFα induced by wild type Salmonella.

As demonstrated in FIG. 4, YS8211 induced TNFα significantly less thanWT 14028. Thus, as shown in FIG. 4, the msbB⁻ strain induced TNFα about33% (i.e., 3 times less) of the wild type msbB⁺ strain.

7.3.2 TNFα Induction in Pigs

An msbB⁻ strain of Salmonella, YS8212, and WT 14028, were first grown tosaturation in LB media at 37° C. with shaking at 225 rpm. A 1:100dilution of these bacterial strains were then transferred to fresh LBand grown to an OD₆₀₀=0.8 at 37° C. with 225 rpm. The bacteria werewashed in phosphate buffered saline and 1.0×10⁹ c.f.u. (about 1×10⁸c.f.u./kg) were injected into the ear vein of Sinclair swine(n=6/strain). After 1.5 and 6.0 hours, serum was harvested, centrifugedto remove the cellular content, and frozen for later analysis. Analysisfor TNFα utilized a Genzyme Predicta ELISA plate, which was read using aGilson spectrophotometer.

Results are presented in FIG. 5 and are expressed as picograms ofTNFα/ml serum.

As demonstrated in FIG. 5, at 90 minutes the level of TNFα induced bythe msbB⁻ strain was significantly lower than that induced by theSalmonella WT 14028.

7.3.3 Salmonella LPS-Induced Respiration in Pigs

Lipopolysaccharide (LPS) from Salmonella WT 14028 and the msbB⁻ clone,YS8212 was prepared using the procedure described by Galanos et al.(1969 Eur. J. Biochem. 9: 245-249). Briefly, LPS was extracted frombacteria which had been grown to OD₆₀₀ of 1.0. The bacteria werepelleted by centrifugation, washed twice with distilled water and frozenat −20 C. LPS was purified by extraction with a mixture of 18.3 mlH20:15 ml phenol in a shaking water bath for 1 hr at 70 C. The mixturewas cooled on ice, centrifuged at 20,000×g for 15 min, and the aqueousphase was removed. LPS was precipitated from the aqueous phase byaddition of NaCl to 0.05 M and 2 volumes ethanol and incubation on ice,followed by centrifugation of 2000×g for 10 min. The precipitation wasrepeated after redissolving the pellet in 0.05 M NaCl, and the pelletlyophilized. The LPS was dissolved in sterile distilled water, andeither 5 μg/kg or 500 μg/kg LPS was injected into the ear vein ofSinclair swine which had been anesthetized with Isoflurane. After 1.5and 6.0 hours, respiration rate was determined and recorded.

Results are presented in FIG. 6 and are expressed as a percentage ofrespiration at time zero (t_(o)).

As demonstrated in FIG. 6, respiration was significantly higher in thepigs administered wild type LPS as compared to those administered theLPS from the msbB⁻ strain. Thus, disruption of the msbB gene inSalmonella, produces a modification in lipid A which results in reducedability to increase respiration.

7.3.4 TNFα Induction in Human Monocytes

Human monocytes were prepared from peripheral blood by centrifugationthrough Isolymph (Pharmacia) and allowed to adhere to 24 well platescontaining RPMI 1640. Salmonella WT 14028 and several of the msbB⁻ 14028strains (YS8211, YS8212, YS8658, and YS1170) were first grown tosaturation in LB media at 37° C. with shaking at 225 rpm. A 1:100dilution of these bacterial strains was then transferred to fresh LB andgrown to an OD₆₀₀=0.8 at 37° C. with 225 rpm. The bacteria were added tothe cell culture wells and the culture medium was harvested after 2.0hours, centrifuged to remove the cellular content, and analyzed for TNFαusing a Genzyme Predicta ELISA plate, which was read using a Gilsonspectrophotometer.

The data are presented in FIG. 7 and expressed as picograms of TNFα/mlserum.

As demonstrated in FIG. 7, the msbB⁻ strains induced TNFα significantlyless than did the wild type strain.

7.3.5 msbB−Salmonella LPS TNFα Induction in Human Monocytes

Human monocytes were prepared from peripheral blood by centrifugationthrough Isolymph (Pharmacia) and allowed to adhere to 24 well platescontaining RPMI 1640.

Lipopolysaccharide (LPS) of wild type and of a number of msbB⁻ mutantSalmonella, (i.e., YS8211, YS8212, YS8658 and YS1170) was prepared usingthe procedure described by Galanos et al. (1969 Eur. J. Biochem. 9:245-249) (see Section 7.3.3 for a brief description). The LPS wasdissolved in sterile distilled water, and quantities ranging from 0.001to 100 ng/ml LPS were added to the cell culture wells. After 15 hoursthe culture medium was harvested, centrifuged to remove the cellularcontent, and analyzed for TNFα using a Genzyme Predicta ELISA plate,which was read using a Gilson spectrophotometer.

The data are presented in FIG. 8 and are expressed as picograms ofTNFα/ml serum.

As demonstrated in FIG. 8, LPS purified from the msbB⁻ strains inducedTNFα significantly less than did the LPS from the wild type strain.

7.4 Disruption of Salmonella msbB Reduces Virulence 7.4.1 In Mice

A culture of wild type Salmonella 14028 and one of its msbB⁻ Salmonellaclones, YS862, were grown in LB medium lacking sodium chloride at 37° C.with shaking at 250 rpm until the cultures reached an OD₆₀₀ of 0.8. Thebacteria were diluted into phosphate buffered saline (PBS) at a ratio of1:10 and the equivalent of 2×10⁷ c.f.u. were injected i.p. into C57BL/6mice bearing B16F10 melanomas. Survival was determined daily, or at twoto four day intervals.

Results are presented in FIG. 9A and are expressed as percent survival.

As shown in FIG. 9A, WT 14028-killed all the mice in 4 days, whereas themsbB⁻ mutant spared 90% of the mice past 20 days, demonstrating asignificant reduction in virulence by the msbB⁻ mutant.

7.4.2 In Pigs

A culture of WT 14028 and one of its msbB⁻ Salmonella clones, YS8212,were grown in LB medium lacking sodium chloride at 37° C. with shakingof 250 RPM until the cultures reached an OD₆₀₀ of 0.8. The bacteria werewashed in phosphate buffered saline and 1.0×10⁹ were injected into theear vein of Sinclair swine (n=4/strain). Survival was determined daily,or at two to four day intervals.

Results are presented in FIG. 9B and are expressed as percent survival.

As shown in FIG. 9B, WT 14028 killed all the swine in 3 days, whereasthe msbB mutant spared 100% of the mice past 20 days, demonstrating asignificant reduction in virulence.

7.5 Tumor Targeting of msbB⁻ Clones

Salmonella WT 14028 with the msbB⁻ modification, were assayed todetermine if they possessed tumor targeting ability using the B16F10melanoma subcutaneous animal model. The msbB⁻ clone, YS8211, was grownin LB media lacking sodium chloride at 37° C. with shaking at 250 rpm toan OD₆₀₀ of 0.8. An aliquot of 2.0×10⁶ c.f.u. was injected i.v. intoC57BL/6 mice which had been implanted with 2×10⁵ B16 melanoma cells 16days prior to the bacterial infection. At two days and five days postbacterial infection, mice were sacrificed and tumors and livers assayedfor the presence of the bacteria by homogenization and plating of serialdilutions.

Results are presented in FIG. 10 and are expressed as c.f.u. bacteria/gtissue. As demonstrated in FIG. 10, a positive ratio of tumor to liver(700:1) was found at 2 days, and increased to a positive ratio of 2000:1at 5 days. Thus, the msbB⁻ mutant maintained the ability to target to asolid cancer tumor.

7.6 Use of Salmonella with Disrupted msbB for Anti-Tumor Activity inVivo

Salmonella typhimurium 14028 msbB⁻ clones YS8211, YS8212, YS7216, andYS1629 and WT 14028 (control) were grown in LB media lacking sodiumchloride at 37° C. with shaking at 250 rpm to an OD₆₀₀ of 0.8. Analiquot of 2.0×10⁶ c.f.u. was injected i.p. into C57BL/6 mice which hadbeen implanted with 2×10⁵ B16 melanoma cells 8 days prior to thebacterial infection. Tumor volume was monitored over time.

Results are presented in FIG. 11. Two of the strains, YS8211 and YS1629,showed significant tumor retardation, i.e., tumor growth inhibition.

7.7 Increased Sensitivity to Chelating Agents

In order to assess the sensitivity of bacterial strains to chelatingagents, bacteria with or without the msbB mutation were grown in thepresence or absence of 1 mM EDTA or 10 mM sodium citrate in Luria Broth(LB) lacking sodium chloride. An overnight culture of each of thebacterial strains was diluted 1 to 100 in fresh media, and grown at 37°C. with shaking at 250 rpm. The effect on growth was determined byspectrophotometric readings at an OD₆₀₀.

WT 14028 and msbB⁻ clone YS8211 were grown in the presence or absence of1 mM EDTA (FIG. 12A). EDTA did not inhibit the growth of WT 14028. Incontrast, the msbB⁻ clone showed near complete cessation of growth after3 hours in the presence of EDTA.

WT 14028 and msbB⁻ clone YS862 were grown in the presence and absence of10 mM sodium citrate (FIG. 12B). The msbB⁺ WT 14028 strain showed littleinhibition by sodium citrate compared to the msbB⁻ strain which showednear complete cessation of growth after 3 hours in the presence ofsodium citrate.

Thus, the msbB⁻ Salmonella mutants exhibited sensitivity to chelatingagents which promote eradication of the bacteria, a characteristic whichis similar to an antibiotic effect. It is envisioned that such acharacteristic would be advantageous for use of msbB⁻ Salmonella mutantsfor in vivo therapy.

In order to further assess the sensitivity of Salmonella strains tochelating agents, the hyperinvasive pur strain YS72, its msbB⁻ strain,YS7216, and a derivative of YS7216, YS1629, were grown in the presenceof increasing concentrations of EDTA. A fresh culture of YS72, its msbB⁻strain YS7216 and its faster-growing derivative YS1629 were diluted 1 to100 in fresh, zero salt LB media containing 0, 0.25, 0.5, 1.0 or 2.0 mMEDTA and grown at 37° C. with 225 RPM for 4 hours, and c.f.u. wasdetermined by plating serial dilutions onto LB plates (Table I). Greaterthan 99% inhibition was achieved for the msbB⁻ strain YS7216 atconcentrations of EDTA greater than 0.25 mM and its derivative YS1629was inhibited greater than 90% at 0.5 mM and greater than 99% at 2.0 mM.In contrast, although the YS72 clone exhibited some sensitivity to EDTAit was not inhibited at the 90% level even at 2.0 mM. TABLE I c.f.u. +EDTA {% inhibition} Strain c.f.u. no EDTA [0.25 mM] [0.5 mM] [1.0 mM][2.0 mM] YS72 3.0 × 10⁹ 2.4 × 10⁹ 1.5 × 10⁹ 7.3 × 10⁸ 4.8 × 10⁸ {20%}{50%} {75%} {84%} YS7216 6.3 × 10⁸ 2.1 × 10⁶ 1.1 × 10⁶ 3.2 × 10⁶ 4.3 ×10⁶ {99.6%} {99.8%} {99.4%} {99.3%} YS1629 1.3 × 10⁹ 6.0 × 10⁸ 1.0 × 10⁸2.9 × 10⁷ 7.5 × 10⁶ {54%} {92%} {97%} {99.4%}

7.8 Bacterial Survival within Macrophages

In order to determine the sensitivity of msbB⁻ Salmonella tomacrophages, two types of macrophages were used: (A) bone marrow-derivedmacrophages obtained from the femurs and tibias of C57BL/6 mice, whichwere allowed to replicate by addition of supernatant from the LADMACcell line which secretes macrophage colony stimulating factor (Sklar etal., 1985. J. Cell Physiol. 125:403-412) and (B) J774 cells (a murinemacrophage cell line) obtained from America Type Culture Collection(ATCC). Salmonella strains used were WT 14028 and its msbB⁻ derivativesYS8211 and YS1170. Bacteria were grown to late log phase OD₆₀₀=0.8 and1×10⁶ were allowed to infect a confluent layer of mammalian cells withina 24. well dish for 30 min, after which the extracellular bacteria wereremoved by washing with culture medium and the addition of 50 μg/mlgentamicin (Elsinghorst, 1994, Methods Enzymol. 236:405-420). Bacteriawere counted by plating serial dilutions of the cell layer removed using0.01% deoxycholate, and expressed as the percent initial c.f.u. overtime.

The results are presented in FIG. 13 and expressed as percent c.f.u. pertime. The msbB⁻ strain shows significantly less survival in macrophages.

7.9 LD50 of msbB Derivatives

Spontaneous derivatives of msbB⁻ strains YS8211 and YS7216 were selectedfrom in vitro culture on non-modified LB medium based upon enhancedgrowth characteristics. These bacterial strains were grown to OD₆₀₀ of0.8 and c.f.u. ranging from 1×10² to 1×10⁸ were injected i.v. into thetail vein of C57BL/6 mice. Acute lethality was determined at 3 days, andthe LD₅₀ determined as described by Welkos and O'Brien (Methods inEnzymology 235:29-39, 1994). The results are presented in Table II.Thus, although all the msbB⁻ strains have a reduced ability to induceTNFα (See Section 7.3.5), the results demonstrate that strain YS1170 issignificantly less attenuated than other msbB⁻ strains and therefore notall msbB⁻ strains are useful for providing both reduced TNFα inductionand reduced virulence. TABLE II Strain LD₅₀ WT 14028 1 × 10³ YS8211 4 ×10⁶ YS8212 3.9 × 10⁷   YS1629 1 × 10⁷ YS1170 1 × 10⁶

8. msbB Mutation in Combination with a Biosynthetic Pathway Mutation

In order to assess compatibility with auxotrophic mutations, as measuredby retention of the ability to target and replicate within tumors,combinations of the msbB mutation with auxotrophic mutations weregenerated. msbB⁺ strains were grown in LB broth or LB plates containing1.5% agar at 37° C. msbB⁻ strains were grown in modified LB containing10 g tryptone, 5 g yeast extract, 2 ml 1N CaCl₂ and 2 ml IN MgSO₄ perliter, adjusted to pH 7 using 1N NaOH. For transducing msbB1::tet, LBlacking NaCl was used, with 4 mg/l tetracycline. Liquid cultures wereshaken at 225 rpm. The msbB1::tet was transduced to auxotrophic strainsto generate YS1604 (msbB⁻, pur⁻, hyperinvasive), YS7232 (msbB⁻, purI⁻,hyperinvasive), YS7244 (msbB⁻, purI⁻, aroA⁻ hyperinvasive), YS1482(msbB⁻, purI⁻, purA⁻). For tumor targeting experiments, cells werediluted 1:100 into LB, grown to OD₆₀₀=0.8 to 1.0, washed in phosphatebuffered saline (PBS), resuspended in PBS, and 2×10⁶ were injected intothe tail vein of C57BL/6 mice. At day 7, tumors were excised, weighed,homogenized, and c.f.u. determined by plating serial dilutions ontomodified LB described above.

Results are presented in Table III and are expressed as c.f.u. per gramtumor tissue. Some of the strains, YS8211, YS1604, and YS7232 show highlevels of c.f.u. within the tumors, whereas YS7244 and YS1482 areapproximately 500 to 5000 times less. TABLE III Strain genetic markerc.f.u./gram tumor tissue YS8211 msbB⁻ 3 × 10⁹ YS1604 msbB⁻, pur⁻,hyperinvasive 9 × 10⁹ YS7232 msbB⁻, purI⁻, hyperinvasive 9 × 10⁹ YS7244msbB⁻, purI⁻, aroA⁻ hyperinvasive 5 × 10⁵ YS1482 msbB⁻, purI⁻, purA⁻ 6 ×10⁶

8.1 Generation of the YS1456 Strain Containing Deletions in msbB ANDpurI

The generation of Salmonella strain YS1456 from the wild type Salmonellatyphimurium is outlined in FIG. 15. The wild type Salmonella typhimuriumwas transduced with purI 1757::Tn10 which conferredtetracycline-resistance, resulting in strain YS1451.

Strain YS1451 was then subjected to a Bochner selection to render thestrain tet sensitive and introduce tet^(s) gene and introduce a purIdeletion (Bochner et al. 1980, J. Bacteriol. 143:926-933), yielding thestrain YS1452. Strain YS1452 was tet^(s) and purI⁻. Strain 1452 was thentransduced with msbB1::tet via bacteriophage P22, using strain YS8211(msbB::tet) as the donor. The resulting strain, YS1453, was initiallysensitive to 10 mM ethylene glycol bis((b-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), spontaneously reverted to aEGTA-resistant phenotype. One such revertant, denoted YS1454, wasselected by plating YS1453 on EGTA (2mM in Luria agar).

Strain YS1454 was then transduced with the msbB2(A) b1a sacB chromosomalelement, selecting for ampicillin resistance. This transduction processbrought in a second version of the disrupted msbB gene, denoted msbB2(Δ)as well as the b1a and sacB genes. The b1a gene is responsible for thetranscription of the enzyme β-lactamase, which metabolizes ampicillin,and was used to select for ampicillin resistant transductants. The sacBgene is responsible for the conversion of sucrose into a toxic chemical,levan, that is lethal to the host cells, and was subsequently used toselect for recombinants which lose or have mutations in sacB (seeSection 7.2.1 for improved pre-selection methods with sucrose). Thepresence of the b1a and sacB genes allowed the selection of the amp^(r)and suc^(s) strain (denoted as strain YS1455), which contained both themsbB1::tet and msbB2(Δ) genes.

Strain YS1455 was then plated on Luria Bertani (LB) sucrose to select asuc^(r) amp^(s) tet^(s) derivative to remove msbB1::tet and restoreantibiotic sensitivity. The derivative was denoted as strain YS1456.

In summary YS1456 has deletion mutations in purI and msbB. It is alsotet^(s) amp^(s) and EGTA^(r).

8.2 Generation of the YS1646 Strain Containing Deletions in msbB andpurI

The generation of Salmonella strain YS1646 from the wild type Salmonellatyphimurium (wild type strain ATCC 14028) is outlined in FIG. 16. Thewild type Salmonella typhimurium was mutagenized with nitrosoguanidineand ultraviolet (UV) light and selected for hyperinvasiveness inmelanoma cells. The resistant strain, denoted YS72, were confirmed topossess tumor-hyperinvasiveness pur⁻ and xyl⁻ properties (Pawelek etal., 1997, Caner Res 57: 4537-4544).

To replace the chromosomal purl gene in strain YS72 with a purldeletion, strain YS72 was transduced with the purl 1757::Tn10 gene,which conferred tetracycline-resistance. The donor for the purI1757::Tn10 gene was Salmonella strain TT11 (purI 1757::Tn10). The donorstrain was originally obtained from the Salmonella Genetic Stock Center(Dept. of Biological Science, Univ. Calgary, Calgary, Alberta, CanadaT2N 1N4). Transduction was performed using bacteriophage P22 (mutantHT105/1 int-201). The transductant, denoted YS1641, was isolatedfollowing selection on tetracycline.

Strain YS1641 was then subjected to a Bochner selection to remove thetet gene and introduce a purI gene deletion (Bochner et al., 1980, J.Bacteriol. 143:926-933), yielding strain YS1642. Strain YS1642 wastet^(s) and purI⁻. The selection of a tet⁻-deleted strain allowedfurther genetic modification (e.g., msbB gene disruption, see nextparagraph) using tet gene transduction. Strain YS1642 has a tight purinerequirement due to purI(A), and has been shown to revert to purI⁺ at afrequency of less than 1 in 10¹⁰ cells.

Strain YS1642 was then transduced with msbB1::tet via bacteriophage P22,using strain YS8211 (msbB::tet) as the donor. The DNA sequence for themsbB gene is shown in FIG. 1. The tet gene in the msbB1::tet geneconfers resistance to 5 mg/L of tetracycline. The resulting strain thusobtained was YS1643.

Strain YS1643 was initially sensitive to 10 mM ethylene glycolbis((b-aminoethyl ether)-N,N,N,N′-tetraacetic acid (EGTA), spontaneouslyreverted to a EGTA-resistant phenotype. One such revertant, denotedYS1644, was selected by plating YS1643 on EGTA (2 mM in Luria agar).

Strain YS1644 was then transduced with the msbB2(Δ) b1a sacB chromosomalelement. This transduction process brought in a second version of thedisrupted msbB gene, denoted as msbB2(Δ) as well as the b1a and sacBgenes. The b1a gene is responsible for the transcription of the enzymeβ-lactamase, which metabolizes ampicillin, and was subsequently used toselect transductants. The sacB gene is responsible for the conversion ofsucrose into a toxic chemical, levan, that is lethal to the host cells,and was used to select for recombinants. The presence of the b1a andsacB genes allowed the selection of the amp^(r) and suc^(s) strain(denoted as strain YS1645), which contained both the msbB1::tet andmsbB2(Δ) genes.

Strain YS1645 was plated on Luria-Bertani (LB) sucrose to select asuc^(r) amps tet^(s) derivative to remove the msbB::tet gene and restoreantibiotic sensitivity (i.e., a derivative with deletion of msbB1::tetb1a sacB). This derivative was denoted as strain YS1646.

In summary YS1646 has deletion mutations in purI, and msbB. It is alsotet^(s), amp^(s), and EGTA^(r).

8.3 Inhibition of Tumor Growth with YS1646 Strain

Intravenous (IV) administration of YS1646, an attentuated strain ofSalmonella typhimurium, resulted in selective replication within tumors,and concomitant inhibition of tumor growth (see FIG. 17 and Table IV).

In all instances, a staged tumor model was used in which tumors wereallowed to become established following tumor cell inoculation and priorto YS1646 administration. As a result of the ability of YS1646 toreplicate within the tumor, a shallow dose-response relationship overthe effective dose range was determined whereby the extent of tumorinhibition, exerted by low doses of YS1646, approached the level oftumor inhibition achieved at higher doses. This suggested that, even atlow doses, significant clinical efficacy could be achieved as long asthe bacteria reached the tumor and accumulated within the tumor. Dosesbelow 1×10² cfu/mouse gave inconsistent results, possibly due tocompetition between the ability of YS1646 to reach and colonize thetumor vs. the ability of the animals to clear YS1646.

The efficacy of YS1646 was evaluated in mice previously implanted withB16-F10 melanoma. In this study a single IV dose of YS1646 at 10⁴, 10⁵or 10⁶ cfu/mouse significantly reduced tumor size when compared tocontrol treatment, and the degree of tumor size reduction wasdose-related. The efficacy observed with the highest dose of YS1646 wassuperior to that with the positive control, CYTOXAN™ (also known ascyclophosamide), whereas the efficacy with the mid-dose of YS1646 wasequivalent to that with, CYTOXAN™. It is important to note that theefficacy induced by YS1646 was induced by a single IV dose, whereas thatinduced by CYTOXAN™ was multiple IV doses (given weekly, for 3 weeks).The ability of YS1646 to inhibit tumor growth, as a function of dose,was examined over an administered dose range of 1×10⁴ to 1×10⁶cfu/mouse. Each dosage group was comprised of 10 tumor-bearing animals,which were randomized prior to bacteria administration. Mice wereadministered bacteria on Day 7, and tumor volumes were measured on Days10, 13, 17, 20, and 24. For comparison, CYTOXAN™ (cyclophosphamide) wasadministered once per week at a dose of 200 mg/kg, beginning on Day 7 aswell. Mean tumor volumes of each group on Day 24 are presented in TableIV. TABLE IV Inoculum Dose Mean Tumor Percent (cfu/mouse) Volume (mm³) ±S.D. T/C Inhibition  0 4728 ± 804 — 0 10⁴ 1011 ± 375 0.214 78 10⁵  560 ±176 0.118 88 10⁶ 279 ± 91 0.059 94

The differences observed between individual groups were deemedsignificant when analyzed either by the Wilcoxon signed rank testanalysis, or by a two-tailed t-test. As indicated in Table IV,increasing tumor inhibition was observed with increasing dose of YS1646.All doses were found to give significant antitumor activity (T/C of lessthan an equal to 42%), as defined by the Drug Evaluation Branch of theDivision of Cancer Treatment, National Cancer Institute (Bethesda, Md.)(Vendetti, J. M., Preclinical drug evaluation: rationale and methods,Semin. Oncol. 8:349-361; 1981), and doses of 1×10⁵ cfu/mouse gaveresults equivalent to or better than cyclophosphamide. A linearcorrelation between YS1646 dose and tumor inhibition was not observeddue to the ability of YS1646 to replicate preferentially within thetumor, which led to greater than expected potency at lower doses.Intravenous adminstration of YS1646, an attentuated strain of Salmonellatyphimurium, resulted in selective replication within tumors, andconcomitant inhibition of tumor growth. Between inoculum doses of 1×10⁴to 1×10⁶ cfu/mouse, a dose-response for inhibition of tumor growth wasobtained, ranging from 78% to 94% inhibition of tumor growth. At the twohighest inoculum doses, the level of tumor growth inhibition wascomparable to or better than that achieved by optimal treatment withcyclophosphamide.

8.4 Virulence

At a dose of 1×10⁶ cfu/mouse, YS1646 does not cause lethality, incontrast to the parental wide type strain ATCC 14028, which causes 100%mortality at a dose of 1×10² cfu/mouse. This indicates that YS1646 isgreater than 10,000-fold less virulent than the parental wild typestrain. The antitumor efficacy was observed at doses of 10⁴ to 10⁶cfu/mouse, whereas lethality was not observed until the doses were >10⁶cfu/mouse. The dose inducing mortality was 1 to 100-fold greater thanthe dose inducing anti-tumor efficacy (see FIG. 18).

8.5 Antibiotic Suppression of YS1646 Induced Mortality Following LethalInfection

The ability of ampicillin and ciprofoxacin to suppress infection byYS1646 was evaluated by determining the ability of antibiotics toprevent mortality in C57BL/6 mice inoculated with 5×10⁶ cfu (LD₅₀equivalent).

Groups were divided into the following treatment categories: 1)untreated control, 2) ampicillin-treated, 3) ciprofloxacin-treated, and4) ciprofloxacin and ampicillin treated. Antibiotic treatment wasinitiated 3 days following bacteria administration and animals wereobserved daily for appearance and mortality for 14 days. Resultspresented herein demonstrate that use of antibiotic was able to supressmortality following lethal bacterial infections (see FIG. 18).

9. Deposit of Microphages

The following microorganisms were deposited with the American TypeCulture Collection (ATCC), 10801-University Blvd., Manassas, Va.20110-2209, on Sep. 9, 1997, and have been assigned the indicatedAccession numbers: Microorganism ATCC Accession No. YS8211 202026 YS1629202025 YS1170 202024

The following microorganisms were deposited with the American TypeCulture Collection (ATCC), 10801 University Blvd., Manassas, Va.20110-2209, on 25 Aug. 1998, and have been assigned the indicatedAccession numbers: Microorganism ATCC Accession No. YS1646 202165 YS1456202164

The invention claimed and described herein is not to be limited in scopeby the specific embodiments, including but not limited to the depositedmicroorganism embodiments, herein disclosed since these embodiments areintended as illustrations of several aspects of the invention. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

A number of references are cited herein, the entire disclosures of whichare incorporated herein, in their entirety, by reference.

1-37. (canceled)
 38. A method of inhibiting the growth or reducing thevolume of a solid tumor cancer, comprising administering an effectiveamount of a mutant Salmonella sp. to a patient having a solid tumorcancer, wherein said mutant Salmonella sp. comprises a geneticallymodified msbB gene and is capable of targeting a solid tumor cancer whenadministered in vivo.
 39. The method of claim 38, wherein the mutantSalmonella sp. is Salmonella typhi, Salmonella choleraesuis, orSalmonella enteritidis.
 40. The method of claim 38, wherein the mutantSalmonella sp. expresses an altered lipid A molecule.
 41. The method ofclaim 38, wherein the mutant Salmonella sp. induces TNFα expression atabout 5 percent to about 40 percent of that induced by a wild-typeSalmonella sp.
 42. The method of claim 38, wherein the mutant Salmonellasp. induces TNFα expression at about 10 percent to about 35 percent ofthat induced by a wild-type Salmonella sp.
 43. The method of claim 38,wherein a chelating agent inhibits the growth of the mutant Salmonellasp. by about 90 percent compared to the growth of a wild-type Salmonellasp.
 44. The method of claim 38, wherein a chelating agent inhibits thegrowth of the mutant Salmonella sp. by about 99 percent compared to thegrowth of a wild-type Salmonella sp.
 45. The method of claim 38, whereina chelating agent inhibits the growth of the mutant Salmonella sp. bygreater than 99 percent compared to the growth of a wild-type Salmonellasp.
 46. The method of claim 43, wherein the chelating agent isEthylenediaminetetraacetic Acid (EDTA), Ethylene Glycol-bis(β-aminoethylEther) N,N,N′,N′,-Tetraacetic Acid (EGTA), or sodium citrate.
 47. Themethod of claim 38, wherein the mutant Salmonella sp. survives inmacrophages at about 50 percent to about 30 percent of the level ofsurvival of a wild-type Salmonella sp.
 48. The method of claim 38,wherein the mutant Salmonella sp. survives in macrophages at about 30percent to about 10 percent of the level of survival of a wild-typeSalmonella sp.
 49. The method of claim 38, wherein the mutant Salmonellasp. survives in macrophages at about 10 percent to about 1 percent ofthe level of survival of a wild-type Salmonella sp.
 50. The method ofclaim 38, wherein the solid tumor cancer is melanoma, colon carcinoma,lung cancer, liver cancer, kidney cancer, prostate cancer, or breastcancer.
 51. A derivative of a mutant Salmonella sp., comprising agenetically modified msbB gene wherein the mutant Salmonella sp.replicates at physiological temperatures, and wherein said derivativefurther comprises a mutation characterized by altered sensitivity to achelating agent when compared to that of an msbB⁻ mutant Salmonella sp.designated YS8211 having ATCC Accession No.
 202026. 52. The derivativeof claim 51, wherein the chelating agent is EthylenediaminetetraaceticAcid (EDTA), Ethylene Glycol-bis(β-aminoethylEther)N,N,N′,N′,-Tetraacetic Acid (EGTA), or sodium citrate.
 53. Thederivative of claim 51, further comprising one or more geneticallymodified genes to auxotrophy, wherein at least one of said genes toauxotrophy is genetically modified AroA, isoleucine biosynthesis, valinebiosynthesis, phenylalanine biosynthesis, tryptophan biosynthesis,tyrosine biosynthesis, or arginine biosynthesis.
 54. The derivative ofclaim 51 which induces TNFα expression at about 5 percent to about 40percent of that induced by a wild type Salmonella sp.
 55. Apharmaceutical composition, comprising an amount of the derivative ofclaim 51, effective to inhibit the growth or to reduce the volume of asolid tumor cancer, and a pharmaceutically acceptable carrier.
 56. Amutant Salmonella sp., comprising a genetically modified msbB gene and agenetically modified biosynthetic pathway gene in which the biosyntheticpathway mutation confers attenuated virulence.
 57. A pharmaceuticalcomposition, comprising an amount of the mutant Salmonella sp. of claim56, effective to inhibit the growth or to reduce the volume of a solidtumor cancer, and a pharmaceutically acceptable carrier.